[unreadable] The research project summarized below, a collaboration between Drs. Schoolnik and Md. Sirajul Islam, will be performed mainly in Bangladesh at the International Center for Diarrhoeal Diseases Research (ICDDR,B) as an extension of NIH grant RO1 AI43422. Observational field studies performed in Bangladesh and elsewhere indicate that Vibrio cholerae, the agent of Asiatic cholera, is a normal member of natural aquatic habitats. In the rivers and estuaries that compose the Gangetic Delta, V. cholerae is believed to be associated with phyto- and zoo- plankton and to reside in mixed-species biofilms on a variety of surfaces. One such surface, the chitinous exoskeletons of copepods, can be degraded by V. cholerae chitinases, thus providing a source of nitrogen, carbon and energy. Other surfaces, including sand particles in the benthic layer of the estuary, are nutrient-poor. Biofilm bacteria on non-nutritive surfaces of this kind must obtain nutrients from the water column. In turn, the nutrient content and chemical features of the water column are dramatically affected by the monsoon seasonal cycle. As a result, the movement of V. cholerae between these sites in its aquatic reservoir and the human host is believed to be determined, in part, by the effects of the monsoon climate on the physicochemical features of this habitat. To begin to understand how these seasonal changes affect V. cholerae- containing mixed-species biofilms in the Ganges Delta, Drs. Islam and Schoolnik recently completed a one year pilot study (supported as a supplement to the parent grant) that showed the following: (1) V. cholerae resides in mixed species biofilms that form on non-nutritive plastic surfaces submerged in the Ganges Delta near Matlab, Bangladesh; and (2), the species composition of the biofilm changes during the year. These observations and the importance and abundance of chitin as a nutritive surface led to the following hypothesis: the population structure of V. cholerae-containing mixed-species biofilms varies as a function of the surface substratum (chitin compared to non-nutritive surfaces) and season (pre-monsoon, monsoon or post-monsoon). To test this hypothesis, the work proposed here will .use a culture-independent, 16S rRNA sequence-based method to determine the population structure of biofilm consortia on chitin or polycarbonate surfaces submerged at the Matlab sampling site during the pre-monsoon, monsoon .and post-monsoon periods. The identified species-specific sequences will then be used to design labeled pligonucleotide probes for fluorescence in situ hybridization (FISH) assays; these probes will be used in the last two years of the project to determine the population structure of monthly biofilm samples from chitin and polycarbonate surfaces. These results will be correlated with changes in water chemistry and cholera case rates. The transfer of each component of these key molecular ecology methods to the ICDDR,B--achieved by a training program for Bangladeshi scientists at the ICDDR,B and at Stanford--will significantly enhance the research capacity of this important Center. [unreadable] [unreadable]